Knockdown of c-MYC Controls the Proliferation of Oral Squamous Cell Carcinoma Cells in vitro via Dynamic Regulation of Key Apoptotic Marker Genes

Oral squamous cell carcinoma (OSCC) is the most common malignant epithelial cancer occurring in the oral cavity, where it accounts for nearly 90% of all oral cavity neoplasms. The c-MYC transcription factor plays an important role in the control of programmed cell death, normal-to-malignant cellular transformation, and progression of the cell cycle. However, the role of c-MYC in controlling the proliferation of OSCC cells is not well known. In this study, c-MYC gene was silenced in OSCC cells (ORL-136T), and molecular and cellular responses were screened. To identify the pathway through which cell death occurred, cytotoxicity, colony formation, western blotting, caspase-3, and RT-qPCR analyzes were performed. Results indicated that knockdown of c-MYC has resulted in a significant decrease in the cell viability and c-MYC protein synthesis. Furthermore, caspase-3 was shown to be upregulated leading to apoptosis via the intrinsic pathway. In response to c-MYC knockdown, eight cell proliferation-associated genes showed variable expression profiles: c-MYC (-21.2), p21 (-2.5), CCNA1(1.8), BCL2 (-1.4), p53(-3.7), BAX(1.1), and CYCS (19.3). p27 expression was dramatically decreased in c-MYC-silenced cells in comparison with control, and this might indicate that the relative absence of c-MYC triggered intrinsic apoptosis in OSCC cells via p27 and CYCS.

ancer in its broad sense is one of the serious diseases with thousands of deaths worldwide recorded each year (1). Several etiology factors underlie cancer, including bad nutrition habits (2), lack of exercise (3), family history (4), and prolonged exposure to environmental methylationmodulating agents (5)(6)(7).
Head and neck squamous cell carcinoma (HNSCC), including oral cancer, is a widespread malignancy with more than 500,000 newlydiagnosed cases per year worldwide (8,9). Oral cancer is one of the most common malignancies not only in the developing, but also in the developed countries, with more than 405,000 new cases reported each year (10)(11)(12). One major type of oral cancer is the squamous cell carcinoma, which is considered the most prevailed histological form accounting for more than 90% of all HNSCC cases (13).
Etiologic factors for oral squamous cell carcinoma (OSCC) include, but are not limited to, alcohol (14) and tobacco (15), where alcohol and tobacco appear to have a synergistic effect in the etiology of OSCC. Other etiologic factors include red meat and salted meat consumption (16), dietary deficiencies (17), and poor oral hygiene (18). These factors cumulatively induce multi-step carcinogenesis process leading to the accumulation of several genetic (oncogenes and tumor suppressers) and epigenetic (hyper-and/or hypomethylation) mutations (19)(20)(21). Recent researches have focused on finding molecular diagnostic/prognostic markers that could help in assigning patients in the right category.
Avian myelocytomatosis virus oncogene cellular homolog (c-MYC) is a member of the MYC family of transcription factors, where it plays an essential role in controlling cell cycle progression (22), programmed cell death (23) and normal-to-malignant cellular transformation (24).
Over expression of the c-MYC gene is observed in different types of cancer including HNSCC (25). c-MYC was also involved in the regulation of telomerase transcription (a major player in the carcinogenesis process) in association with different E26 transformation-specific (Ets) transcription factor family members (26). p53 (27) and p16 (28) are among the critical tumor suppressor genes that are highly studied in OSCC, with p53 being mutated in about 90% of HNSCC cases (29,30).
In the present study, it was aimed to determine the role of c-MYC in controlling the proliferation of

Caspase-3 assay
To find the activity of caspase-3 enzyme, a colorimetric Caspase 3 Assay Kit (Sigma-Aldrich, USA) was used according to the recommendations of the manufacturer. The assay was performed in 1 ml reaction mixture, and the absorbance was read by using a plate-reader (Biotek, Neo2) at 405 nm.

RNA extraction and cDNA synthesis
Total RNA was extracted from (1)

Gene expression analysis
RT-qPCR was used to amplify some marker genes associated with intrinsic apoptotic pathways, oncogenic pathway, and cell cycle control BAX, p27, and cytochrome C, somatic (CYCS)).
Primers sequences are presented in Table 1
Also, it is found that spontaneous apoptosis in p27positive tumors is higher than that in p27-negative OSCC (49). In this study, the suppression led to apoptosis as revealed by cytotoxicity assay and western blotting. It is indicated that knocking down